Seropositivity among Blood Samples Drawn from Suspected Dengue Cases at a Tertiary Care Centre of Nepal: A Descriptive Cross-sectional Study

ABSTRACT Introduction: The cases of dengue fever have been reported more frequently in Nepal these days. The aim of this study was to find the prevalence of seropositivity among blood samples drawn from suspected dengue cases at a tertiary care centre of Nepal. Methods: A descriptive cross-sectional study was conducted at a tertiary care hospital from 1st June 2017 to 31st October 2018 after getting approval from the Institutional Review Committee (Reference number: 23/2016). A total of 537 suspected dengue patients were selected for the study using convenience sampling. These dengue positive sera were assayed for their reactivity with Immunoglobulin M and Immunoglobulin G present in sera and synthetic peptides of dengue virus by enzyme linked immunosorbent assay. Data was entered and analysed in Microsoft Excel 2016. Point estimate at 95% Confidence Interval was calculated along with frequency, percentage, mean and standard deviation. Results: Among 537 suspected dengue cases, the seropositivity for dengue was found in 124 (23.09%) (19.52-26.65 at 95% Confidence Interval) of the serum. Conclusions: The present study revealed that dengue was more prevalent in our setting compared to similar studies. All the synthetic peptides showed reactivity with dengue-positive sera with maximum reactivity shown by RR2 peptide. In dengue-positive sera, RR2 peptide of dengue virus identified more Immunoglobulin M than Immunoglobulin G.


INTRODUCTION
Dengue fever is a viral disease spread by mosquito of Flaviviridae family. 1,2 Antigenically, four different serotypes of the dengue virus with 65% genomic similarity exist. 3,4 Commercially available kits use mixes of inactivated viral preparations or recombinant envelope proteins, so detection of anti-dengue antibodies is problematic. 5 The envelope (E) protein and non-structural protein (NS1) encoded by dengue virus are very important in the pathogenicity of dengue virus infection and are highly conserved in all strains. 6 Hence, these two proteins were selected to identify epitopes for detecting anti IgM and IgG antibodies. These epitopes were synthesised as linear peptides with the help of DNA star approach. 7 The aim of this study was to find the prevalence of seropositivity among blood samples drawn from suspected dengue cases.

METHODS
This was a descriptive cross-sectional study carried out at a tertiary care hospital from 1 st June 2017 to 31 st October 2018 after getting approval from the Institutional Review Committee (Reference number: 23/2016) of Nobel Medical College and Teaching Hospital (NMCTH). All the patients, who presented to the emergency and medicine departments of NMCTH, suspected with dengue were the study population. Convenience sampling was done. The sample size was calculated by using the formula: n= Z 2 x p x q / e 2 = (1.96) 2 x 0.5 x (1-0.5) / 0.05 2 = 385 Where, n= minimum required sample size Z= 1.96 at 95% Confidence Interval (CI) p= prevalence taken as 50% for maximum sample size q= 1-p e= margin of error, 5% The sample size calculated was 385. However, we have included 537 patient's samples from Clinical laboratory services, NMCTH. Dengue-positive serum was diagnosed based on the NS1 antigen in an immunochromatographic card test kit. These denguepositive sera were assayed for their reactivity with IgM and IgG present in the sera and synthetic peptides (RR2, RR3, RR4, RR5, RR6, RR8) of dengue virus by ELISA technique.
At PH 9.6, 400ng of peptide was dissolved in 100ml of 0.05M carbonate bicarbonate buffer. At 37°C for 4 hours, the dissolved peptide was coated in the wells of microtiter plates. The wells were rinsed three times with PBS/0.05% Tween 20 (PBS-T) before being blocked overnight at 4°C with 200ml blocking solution (five percent skimmed milk powder in PBS-T). After washing the plates, Chikungunya (CHIKV), DENV, and negative sera (1:100 dilutions) were added to duplicate wells and incubated at 37°C for two hours. Antigen-antibody complexes were identified using HRP-conjugated goat anti human IgM/IgG antibodies (1:2000). The plates were cleaned as before, and colour was developed with a TMB-containing substrate solution. Adding 100 micro litre of 1N sulphuric acid and optically stopping the reaction and optical density (OD) was measured at 492nm. The synthetic peptide RR2 was tested for nondengue viral reactivity using CHIKV sera. The sera that were neither IgM nor IgG DNEV positive were used as a negative control.
To test the diagnostic efficiency of peptide with patient sera, a cutoff value for optical density (OD) was calculated. The cutoff values for peptide were in the range of 0.31-0.34 based on the seroreactivity of 18 healthy human sera tested for IgM. We looked at the seroreactivity of all dengue positive and 20 chikungunya positive patient sera based on the cutoff value for this peptide. The computed cutoff value for IgG was in the range of 0.33-0.38. There was no seroreactivity with any of the peptides in any of the 20 chikungunya positive and healthy sera. When the OD Value of a human blood sample was found to be greater than the cut-off value when tested for IgM and IgG in an ELISA, it was declared reactive with synthetic peptides.
All the peptides were synthesised, purified and authenticated in the All India Institute of Medical Sciences (AIIMS), New Delhi. The Peptide was selected on hydrophilicity, secondary structures, antigenicity index, amphipathicity, using Bcepred/DNA star software for B cell and T cell prediction. These peptides were synthesised by Fluorenylmethoxycarbonyl Chemistry. 7 The readymade form of these synthetic peptides were gifted to us by AIIMS to carry out the study in Nepal.
The data was entered and analysed in Microsoft Excel 2016. Point estimate at 95% Confidence Interval was calculated along with frequency, percentage, mean and standard deviation.

RESULTS
Among 537 suspected dengue cases, the seropositivity for dengue was found in 124 (23.09%) (19.52-26.65 at 95% Confidence Interval) of the serum. Out of 124 dengue positive patients, 71 (57.3%) were male and 53 (42.7%) were female. The mean age of dengue positive patients was 41±19. 6 year with a range from 5 to 83 years. The maximum number of dengue positive patients was 47 (37.9%) in the 21-40 years age group. Thirty-six (29.03%) of patients were dengue positive in the 41-60 years age group (Table 1).  The samples having OD more than the cut-off values were considered reactive. With RR2 peptide the dengue positive sera, the maximum OD obtained was 3.3 and the minimum OD value was 0.5 ( Figure  1). Similarly, the IgG dengue positive sera were also reacted with RR2 peptide and found that 60 samples were considered reactive. The maximum OD value obtained was 2.4 and minimum was 0.46 ( Figure 2). The data for each sera OD when reacted with other peptides while detecting IgM or IgG are not shown.

Dengue viral infection is a serious health concern.
Dengue infection is difficult to control because it necessitates effective vector control as well as early, precise, and strain-specific diagnosis. Dengue fever diagnosis has remained a challenge for disease treatment, particularly in developing countries like Nepal. Therefore, we have made an attempt to study the sero-reactivity of synthetic antigenic peptide of dengue virus with dengue positive sera for the first time in Nepal. This peptide can be used as a tool for the diagnosis of dengue in future. The seropositivity was found in 23.09% of the serum in the present study. Rijal  reported in his study that NS1 specific IgG, IgM and IgA antibodies are found in the sera of patients with dengue fever and dengue hemorrhagic fever. 13 In many of the studies, these chemically synthesised peptides have been studied as a alternate source of antigen for diagnosis. 14 RR2 synthetic peptide of dengue virus was reactive with dengue positive sera in the present study, which makes it suitable for the easy and accurate diagnosis of dengue infection in developing country like Nepal, where dengue is endemic. The study also demonstrates that such synthetic peptide can be used easily for dengue diagnosis in the set up of hospitals in countries like Nepal.
There are some limitations of the present study. Dengue-positive patients had been included from one study centre. The results would have been interpreted more reasonably if the more dengue-positive samples would have been enrolled from different epidemic regions of Nepal.